Summary ThelongtermgoalsoftheprojectaretoidentifythefullarrayofeffluxpumpsofStaphylococcusaureus thatcontributetomultipleantimicrobialresistanceandtoelucidatethedeterminantsoftheirexpression, theirroleinmicrobialphysiologyandtheireffectonbacterialresponsetoantimicrobialsininfection.The work will focus on genetic analysis of regulatory elements and on bacterial fitness and response to antimicrobials in a subcutaneous abscess model, collaborating with other project groups to assess the efficacyofnovelantimicrobialcompoundsinabscessesandtheextenttowhicheffluxpumpsaffectthat efficacy.Therearefourspecificaims:1)analyzetheglobalarrayofeffluxpumpsofS.aureusfortheir effects on susceptibility to established antimicrobials and novel compounds identified by P01 collaborators;2)analyzetheeffectsofphysiologicpumpoverexpressionintheabscessenvironmentand on treatment response to antimicrobials with focus on the Tet38 pump and tetracycline treatment; 3) dissect the regulatory networks affecting resistance effux pump expression using the high?efficiency multiplexlibrariesdevelopedbytheWalkerlab;and4)testnovelcompoundsfromP01collaboratorsfor efficacy in mammalian infection and biofilm models and assess the moonlighting model in the abscess model.TheworkwillutilizegeneticmanipulationandallelicexchangeinS.aureus,measurementsofgene expression with RT?PCR, and established murine models of infection (subcutaneous abscess, renal abscess,lethality)utilizingagenomicallydefinedstrainsofmethicillin?resistantandotherS.aureus.The overall goal of the program project is to take a well?integrated, multi?disciplinary approach to understandingantibioticresistancedevelopmentandtransmission,andtointegratethateffortwiththe search for compounds that compromise resistant pathogens, including methicillin?resistant S. aureus (MRSA), by inhibiting novel targets and pathways. This project will add to understanding of resistance mechanismsrelatedtomultidrugeffluxpumpsandprovidestrainsfortestingtheeffectofsuchpumps onnovelcompoundsactiveagainstnewtargetsandpathways.Itwillalsoutilizemammalianmodelsofa commonMRSAinfectiontotestcompoundactivityinvivo.